Effect of 5-lipoxygenase and cyclooxygenase blockade on porcine hemodynamics during continous infusion of platelet-activating factor

PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS

Effect of SLipoxygenase and Cyclooxygenase Blockade on Porcine Hemodynamics During Continuous Infusion of Platelet-Activating Factor N. C. Olson. K. T. Kruse-Elliott

and L. W. Johnson

Department of Anatomy. Physiological Sciences and Radiology. College of Veterinary Medicine. North Ccrrolina Stcr!e Urriversity, 4700 Hillsborough Street, Raleigh, NC 27606. USA (Reprint request to NCO) ABSTRACT. We hypothesized that S-lipoxygenase and cyclooxygenase products might he mediators of cardiopulmonary and systemic vascular effects induced by a 4 h continuous infusion of platelet-activating factor (PAF, 10 ng/kg/min) in anesthetized pigs. Indomethacin (cyclooxygenase inhibitor) potentiated and CGS 8515 (5lipoxygenase inhibitor) attenuated PAF-induced increases in total peripheral resistance (TPR) from 2.5 to 4 h. However, the S-lipoxygenase inhibitor failed to modify pulmonary vasoconstriction and hypertension caused by PAF. Except for a delay in onset (-44 s) and rate of development of pulmonary hypertension during the initial 10 min of PAF infusion, the pulmonary hemodynamic changes were also not attenuated by indomethacin. On the other hand, at 4 h, the PAF-induced pulmonary hypertension and systemic vasoconstriction were completely or partially reversed, respectively, by WEB 2086 (PAF receptor antagonist). The PAF-induced increases in plasma thromboxane B, (TXB,) were blocked by indomethacin but not by CGS 8515, and at 4 h the 5-lipoxygenase inhibitor potentiated the levels of TXBz in pigs treated with PAF. The plasma concentrations of 6-keto-PGF,, and leukotriene B, (LTBJ were not modified by PAF or CGS 8515 + PAF. We conclude that PAF-induced increases in TPR (2.5-4 h) are potentiated by indomethacin and are dependent on Mipoxygenase products other than LTB4. Although the early pulmonary vascular response (~10 mitt) to PAF is dependent on cyclooxygenase products, the sustained response (after 10 mitt) cannot be explained by either 5-lipoxygenase or cyclooxygenase products but may be mediated directly by PAF receptors.

INTRODUCTION

sensitivity reactions, endotoxemia, and adult respiratory distress syndrome (2-4). We and others (S-12) have previously reported the effects of an intravenous (IV) bolus of PAF on the porcine cardiopulmonary system before and after indomethacin (cyclooxygenase inhibitor). or after treatment with the PAF receptor antagonist SRI 63-675 (reviewed in reference 3). Both indomethacin and SRI 63-675 blocked the cardiopulmonary effects induced by bolus administration of low doses of PAF (2-20 ng/kg, IV) (3). Similarly, OKY-046 (TXA, synthase inhibitor) and SQ 29548 (TXA, receptor antagonist) blocked or markedly attenuated pulmonary hypertension induced by PAF (bolus) in anesthetized pigs (S-7. 1I, 12). Moreover, LY 171883 (LTD,/LTE, receptor antagonist) failed to modify the hemodynamic responses induced by an IV bolus of PAF (5). Thus, taken together. these findings provide strong support that the acute porcine hemodynamic response to a bolus infusion of PAF is mediated through secondary release of cyclooxygenase products, particularly TXA, (3, 5-l 2 ). On the other hand, it has been suggested that mechanisms relating to actions of PAF may be dependent on

Platelet-activating factor (PAF), 1-0-alkyl-2-acetyl-snglycero-3-phosphocholine, is a biologically active etherlinked phospholipid that can be synthesized by macrophages, neutrophils, platelets, basophils, eosinophils. mast cells, and endothelial cells (1, 2). PAF has been reported to have numerous biological effects that include alterations in systemic and pulmonary hemodynamics, bronchoconstriction, activation of platelets and leukocytes. increased vascular permeability, edema, and modulation of cellular immune responses (2, 3). Although PAF may elicit these effects directly, many responses may be mediated indirectly through release of secondary mediators, e.g. thromboxane A? (TXA,), prostaglandins (PGs), and leukotrienes (LTs) ( 1, 4). Because PAF has potent and diverse biological activity, it may be an important mediator of a variety of inflammatory conditions, including asthma, anaphylaxis, hyper-

Date received 23 December I992 Date accepted 8 March 1993 549

550

Prostaglandins

Leukotrienes

and Essential Fatty Acids

the manner in which the autocoid is tested (11). If this hypothesis is correct, it could have important implications with regard to therapy. For example, clinical situations in which release of PAF might be intermittent and of short duration would be expected to respond effectively to cyclooxygenase inhibitors. In fact, the porcine cardiovascular response to a bolus infusion of PAF would support such therapy. In contrast, pathophysiological syndromes associated with more sustained release of PAF might be relatively refractory to cyclooxygenase inhibitors. Indeed, in a preliminary experiment, we observed marked and sustained cardiopulmonary alterations within 2 min after the onset of a continuous infusion of PAF (10 ng/kg/min) in an indomethacintreated pig, indicating that the sustained response was independent of the cyclooxygenase pathway. These findings suggest that the signal transduction pathway(s) employed by PAF may be importantly dependent on the infusion protocol as well as duration that the cardiovascular system is exposed to PAF. Thus, in evaluating potential therapies for prolonged exposure of PAF to the cardiopulmonary system, a sustained infusion of the autocoid would seem more appropriate. In the presence of specific inhibitors of 5-lipoxygenase and cyclooxygenase, the hemodynamic effects to a prolonged infusion (i.e. 4 h) of PAF has not been reported in pigs. In the present investigation, we hypothesized that the cardiovascular response to a sustained infusion of PAF might be mediated directly by the autocoid, or possibly indirectly through secondary release of 5lipoxygenase products. To address our hypothesis we utilized the PAF receptor antagonist WEB 2086 (3) and specific inhibitors of the 5-lipoxygenase (CGS 8515) (13, 14) and cyclooxygenase (indomethacin) pathways while maintaining a continuous infusion of PAF.

METHODS Porcine preparation Domestic pigs, 10-16 weeks old weighing 23.1 f 0.8 kg, were anesthetized (25 mg/kg, IV) and maintained (6 8 mg/kg/h, IV) with pentobarbital sodium (Butler). After the pigs were placed in supine position, a tracheotomy was performed and a cuffed endotracheal tube was inserted into the trachea. The pigs were mechanically ventilated (Harvard respirator) on room air at a constant tidal volume (10-15 ml/kg). Respiratory rate (14-18 breaths/min) was initially adjusted to an end-tidal PC02 of -34 mmHg (Hewlett-Packard capnometer). Once the respiratory parameters were established for each animal they were held constant for the remainder of the experiment. Polyethylene catheters were inserted into a carotid artery and both internal jugular veins for blood collection and drug administration, respectively. A 7-Fr triplelumen Swan-Ganz catheter and a 5-Fr thermistor-tip catheter (Baxter) were inserted into the pulmonary artery (via the femoral vein) and femoral artery, respectively.

The catheters were connected to Statham P23 Gb pressure transducers for measurements of mean pulmonary arterial (P,,), pulmonary arterial wedge (P,,). and mean aortic pressures. Heparin (5000 IU, LyphoMed) was administered IV to maintain catheter patency. Pressure recordings were made continuously on a multichannel Gould recorder. Cardiac output was measured by the thermal-dilution technique (model 93 10 cardiac output computer, Baxter) at 0, 0.17, and 0.5 h, and thereafter at 0.5 h intervals until 4 h. Cardiac output was normalized for body weight and expressed as cardiac index (CI) in ml/s/kg. Pulmonary vascular resistance (PVR) and total peripheral resistances (TPR) were calculated as: PVR = (P,,-P,,,)/CI, and TPR = P,$CI, respectively. A Coulter counter determined the platelet and total white blood cell (WBC) count at 0, 1, and 10 min and at 1, 2, 3, and 4 h. A differential leukocyte count was determined from blood smears treated with Wright’s stain. At the conclusion of each experiment, the pigs were euthanized with pentobarbital sodium.

Experimental protocols Four groups of pigs were evaluated: Groups 14 were treated with lyso PAF (control, n = 6), PAF (n = 7), indomethacin + PAF (n = 7), and CGS 8515 + PAF (n = 7) respectively. Lyso PAF and PAF were administered at 10 nglkglmin for 4 h. The dose of PAF was based on preliminary experiments that indicated sustained and reproducible cardiovascular changes would be achieved. PAF was prepared by drying chloroform solutions of l-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (Sigma Chemical Co.) under a stream of N2 and resuspending in saline + 0.25% bovine serum albumin (BSA). Lyso PAF (Sigma Chemical Co.) was also suspended in 0.25% BSA. Indomethacin (Sigma Chemical Co.) was infused at 5 mg/kg from -0.5 to 0 h + 3 mg/kg/h from o-4 h. Indomethacin (Sigma Chemical Co.) was prepared by dissolving the calculated dose, for each pig, in 60-100 ml phosphate buffered saline (PBS) (alkalinized with 1 N NaOH to pH -8). Because CGS 8515 is water insoluble, the calculated dose for each pig was initially dissolved in dimethylacetamide (DMA, Sigma Chemical Co., 20 mg CGS 8515/ ml) as previously reported (13-15). The 5-lipoxygenase inhibitor was then diluted, while heating under constant stir, by an equal aliquot of polyethylene glycol400 (PEG 400) to a final concentration of 10 mg CGS 8515/ml of DMA: PEG 400. The infusion syringe and polyethylene tubing containing the CGS 85 15 infusate were covered with a heating pad during the infusion period. CGS 85 15 was infused at 5 mg/kg from -0.5 to 0 h + 2 mg/kg/h from O-4 h. (Due to a technical error, one pig received a maintenance dose of 3.2 mg/kg/h from o-4 h). We previously validated that the loading dose of 5 mg/kg CGS 85 15 blocked calcium ionophore-stimulated LTB, biosynthesis (but not TXB3 in porcine whole blood stimulated ex vivo (15). In one additional pig, CGS 85 15

Effect of 5-Lipoxygenase

and Cyclooxygenase

Blockade on Porcine Hemodynamics

and indomethacin were infused simultaneously through separate catheters. We previously reported that the vehicle for CGS 8515 (DMA:PEG 400) did not alter hemodynamic effects induced by a 6 h infusion of cytokines in anesthetized pigs (I 5). After the 4 h data were collected, an infusion of WEB 2086 (PAF receptor antagonist) was administered at I mg/kg (IV) in groups 2 (n = 3) 3 (n = 3), and 4 (n = 5) to assess for acute reversal of PAF-induced changes on pulmonary and systemic hemodynamics. WEB 2086 (Boehringer-Ingelheim Corp.) was prepared by dissolving the calculated dose in 10 ml saline. After treatment with WEB 2086 (IV over -30 set), Ppa rapidly decreased and reached a steady-state value within 5 min. Cardiac output was again assessed 10 min following administration of WEB 2086 (i.e. at 250 min). The infusion of PAF was mamtained at 10 ng/kg/min during this 10 min period.

Extraction

of LTB,

Aortic blood (10 ml) was collected into siliconized glass tubes containing 3 mg/ml EDTA and 28 pM indomethacin, centrifuged at 1100 g at 4°C for 10 min, and the plasma decanted. I ml of plasma was stored (-80°C) for radioimmunoassay (RIA) of cyclooxygenase products and the remaining plasma was acidified to pH -3.0 with 1 M H,PO,, centrifuged at 1100 g at 4°C for 10 min, and the supematant decanted and extracted as follows: two waters reverse-phase C,s Sep-Paks were placed in series and washed sequentially with 10 ml 100% methanol, 10 ml H20, 10 ml methanol in 0.1% EDTA, and IO ml H,O. 2 ml plasma were then passed through the Sep-Paks, followed by washes with 10 ml H,O and IO ml hexane. To elute LTB,, 10 ml ethyl acetate was passed through the Sep-Paks and the eluate was collected into a siliconized flask. The ethyl acetate eluate was evaporated to total dryness on a rotary evaporator, resuspended in 2 ml 100% methanol, and stored at -80°C until reverse-phase high performance liquid chromatography (RP-HPLC) and RIA could be performed. These extraction procedures were performed at 0, 0.5, 2, and 4 h, with two separate 2 ml aliquots of plasma extracted in tandem and eluted into the same flask. Prior to extraction, PGB, (50 ng/ml plasma) was added to each sample to serve as an internal standard for RP-HPLC.

RP-HPLC Plasma extracts were dried under N, gas and resuspended in 300 ~~160% methanol-H,O. LTB4 was separated by RP-HPLC on an Ultrasphere (Beckman) 5 urn octadecylsilane column (4.6 x 250 mm). LTB4 was eluted using a 60% methanol-H,0 mobile phase (buffered with acetic acid and brought to pH 6.2 with ammonium hydroxide) for 40 min. A Waters C,s precolumn was used to protect the analytical column. The 1 min (1 ml) fractions were collected into separate polypropylene centrifuge tubes and stored at -80°C for later RIA.

During Continuous

Infusion of Platelet-Activating

Factor

55 I

Standards were run daily using a cocktail of unlabeled PGB,, LTD4 and tritiated LTB,. The cold standards were monitored by UV spectrophotometry (Waters LambdaMax 481 displayed on a Waters 740 data module) and the radiolabeled standards by liquid scintillation spectrometry (LKB model 1217 or 1219). Cold standards were selected based on elution times similar to the compound of interest. Elution times from these standard runs were used in selection of the appropriate RP-HPLC fractions to be quantitated by RIA. The column and injector were washed with 100% methanol between RP-HPLC runs to eliminate standard-to-sample or sample-to-sample contamination.

RIA Tritiated TXB,, 6-keto-PGF,,, and LTB, RIA kits were purchased from Amersham Corp. (Arlington Heights, Illinois). For cyclooxygenase products, the RIAs were performed directly on plasma according to instructions provided by the manufacturer. LTB4 was quantitated by RIA of RP-HPLC fractions. Two to three RP-HPLC fractions on either side of the fraction corresponding to the peak elution times (based on the daily standard RPHPLC runs of tritiated LTBJ) was pooled. The methanol component of the pooled fractions was first dried under vacuum in a Speed Vat Concentrator. The remaining aqueous solution was then lyophilized and the residue resuspended in 1 ml 100% methanol. Prior to RIA, an aliquot of this pooled sample was dried and resuspended in assay buffer. Standard curves and analyte concentrations were analyzed by a smooth spline function with weighted points using the LKB 12 19- 102 RIA concentration calculation program. The nonspecific crossreactivities at 50% bound-to-free ratios (B/B,) are very low against all eicosanoids tested. For each RIA kit. the detection limit was 2.5, 3.5, and 0.8 pg/RIA tube for TXB,, 6-keto-PGF,,, and LTB,, respectively. These values were assigned to assay tubes containing analyte concentrations below detectable limits for each respective RIA. The TXB, and LTB, RIAs were performed on samples collected from all pigs (except for LTB+ in lyso PAF-treated pigs where n = 3). The 6-keto-PGF,, RIA was performed only in pigs treated with PAF (n = 4) and CGS 85 15 + PAF (n = 7).

Assessment of TXB, and LTB4 biosynthesis blood stimulated in vitro

in whole

To evaluate the possibility that the 5-lipoxygenase inhibitor CGS 8515 might have non-specific effects on cyclooxygenase and TXA, synthase pathways, we stimulated whole blood with calcium ionophore A23 187 in vitro. A stock solution was made by dissolving 10 mg A231 87 (free acid) in 1 ml dimethyl sulfoxide (DMSO, Sigma Chemical Co.). An aliquot of this solution was diluted 1:5.6 (vol/vol) in distiled HZ0 to avoid hemolysis. In eight pigs (not included in groups lil),

552

Prostaglandins

Leukotrienes

and Essential Fatty Acids

aortic blood was rapidly collected into two 50-ml polypropylene tubes containing heparin (25 U/ml blood). Leukocyte and platelet counts were determined (Coulter counter) and 6 ml aliquots of blood were rapidly pipetted into siliconized glass tubes containing 50 pl DMSO (0.17%, vehicle for A23 187), A23 187 (29 @I; Sigma Chemical Co.), CGS 8515 (0.1-100 PM), or BW755C (100 PM; Burroughs Wellcome Co.). BW755C is a dual inhibitor of the cyclooxygenase and lipoxygenase pathways (13, 14). Blood and CGS 8515 or BW755C were preincubated for 5 min prior to administration of A23187 (29 PM). After 30 min of incubation with A23187 (37°C water bath with a gentle shake; model G76, Gyrotory), 30 ~1 BW755C were added to all tubes (100 PM final concentration) not pretreated with the dual inhibitor. The test-tubes were vortexed and immediately centrifuged (1 100 g, 5 min, O’C). An aliquot (-1 ml) of plasma was decanted and stored at -80°C for TXB2 analysis. 2 ml of plasma were extracted by use of Sep-Paks (n = 6). Then, 500 ~1 extracts from each treatment were combined and the pooled samples subjected to RP-HPLC and RIA as described above.

0 @Zl = I

Control A23187 CGS 8515 + A23167 BW755C + A23167

1800 1600 F

1400

$1200 -H 1000 E

600

z

600

% &

400 200

Statistical analysis Data are expressed as mean or % baseline + SE. Statistical analysis was performed using two-way analysis of variance for data within a treatment group and one-way analysis of variance between groups. Multiple means were compared using Tukey’s omega procedure. Means were considered significantly different at P c 0.05. RESULTS Effect of A23187 and CGS 8515 on plasma TXB, and LTB, concentrations Figure 1 shows that 0.1-100 FM CGS 8515 did not modify A23187-induced increases in TXB, levels in porcine blood (in vitro). On the other hand, 0.1 FM CGS 8515 reduced the A23187-induced increase in LTB4 concentration (i.e. average value present in pooled extracts derived from blood samples of 6 pigs) by 64%. At concentrations 21.0 PM, the 5-lipoxygenase inhibitor blocked A23187-induced increases in LTB,. As expected, BW755C (dual inhibitor of cyclooxygenase and lipoxygenase pathways) blocked A23 187-induced increases in plasma TXB, and LTB4 concentrations. These results are consistent with previous reports which emphasized the selectivity of CGS 85 15 for the 5lipoxygenase pathway (13, 14). Moreover, CGS 8515 potently inhibited LTB4 biosynthesis in guinea pig leukocytes and human whole blood stimulated with A23 187 in vitro (IC,, 0.1 and 0.8 yM, respectively) (13). Effect of lyso PAF, PAF, indomethacin CGS 8515 + PAF on hemodynamics Figure 2 shows that PAF gradually

+ PAF, and

decreased CI to 84 +

b

CGS 6515 O1M)

__fl

BW755C U)

Fig. 1 Effect of CGS 8515 (0.1-100 PM) and BW755C (100 p&l) on plasma levels of (A) TXBz and (B) LTB, after 30 min stimulation of porcine whole blood (37°C) with calcium ionophore (A23187, 29 l.tM). In (A), n = 8 for each treatment (except for 30 PM CGS 85 15 where n = 6). In (B), the Sep-Pak plasma extracts (n = 6) were first pooled and then subjected to RP-HPLC and RIA as described in METHODS. Values are means + SE in (A) and means in (B). Notice that BW755C. a dual inhibitor of cyclooxygenase and lipoxygenase pathways, blocked A23 187-induced increases in TXB, and LTB,. In contrast, l-100 pM CGS 8515 blocked A23187-induced increases in LTB4 but did not modify the increases in TXB2, indicating selectivity for the 5-lipoxygenase pathway. Notice that the ordinate axis is different in (A) versus (B). *P < 0.05 versus control (i.e. DMSO vehicle) value; +P < 0.05 versus A23187 and CGS 8515 + A23187 values.

4 and 7 1 + 4% of the baseline (0 h) value at 1.5 and 4 h, respectively. This was not significantly modified by indomethacin or CGS 85 15, except at 4 h when both drugs caused a small potentiation of the PAF-induced fall in CI. The baseline CI values for pigs treated with lyso PAF, PAF, indomethacin + PAF, and CGS 8515 + PAF were 1.7 + 0.1, 1.7 + 0.1, 1.6 rt 0.1, and 1.8 & 0.1 ml/s/ kg, respectively. Figure 3 shows the increase in TPR as compared to the respective baseline value for each treatment group. From 0.5-4 h PAF caused an increase in ATPR; this increase was significantly potentiated by indomethacin from 2.5-4 h. Conversely, the 5-lipoxygenase inhibitor, CGS 85 15, attenuated PAF-induced systemic vasoconstriction after 2 h (not statistically significant at 3.5 h). Although ATPR increased in control pigs treated with lyso PAF, the increases were of small magnitude. The 0 h TPR values for pigs treated with lyso PAF, PAF, indomethacin + PAF, and CGS 85 15 + PAF were 56 k 5,

Effect of 5Lipoxygenase

120

T

T

I

0

and Cyclooxygenase o-o e-0

LysoPAF PAF

A-A

Indomethacin + PAF CGS 8515 + PAF

u-0

7

Blockade on Porcine Hemodynamics

During Continuous

60-

1

2

3

4

Hour Fig. 2

Effect of a continuous infusion of lyso PAF (n = 6). PAF (n = 7) indomethacin (cyclooxygenase inhibitor) + PAF (n = 7), or CGS 85 15 (5-lipoxygenase inhibitor) + PAF (n = 7) on Cl. PAF and lyso PAF were infused at 10 ng/kg/min. Values represent means + SE and are expressed as % baseline (0 h) value. *P < 0.05 versus the respective baseline value: ‘P < 0.05 versus PAF alone at the respective time interval.

03 + 7, 80 + 9 (P < 0.05 versus other groups), and 52 + 3 mmHg/ml/s/kg, respectively. At 0 h mean aortic pressure in pigs treated with lyso PAF, PAF, indomethacin + PAF, and CGS 8515 + PAF were 92 + 7, 106 i 8, 124 + 8 (P < 0.05 versus other groups), and 93 + 4 mmHg, respectively; at 4 h these values were 101 + IO, 115+9, 116+8,and72+7mmHg(P
Lyso PAP

0-e

PAF Indomethacin + PAF CGS 8515 + PAF

Cl-Cl

<

60--

2

50--

E 2

40--

“E

30--

g

20--

*t

Factor

*t

55.1

*t s-t

T

1

T /,AIA &---A T/ T/f @,._~,&.

0

I

o-o A -A

^M 70--

Infusion of Platelet-Activating

r

1

2

3

4

Hour Fig. 3 Effect of lyso PAF, PAF, indomethacin + PAF. or CGS 85 15 + PAF on the increases in total peripheral resistance (ATPR) as compared to the respective baseline value. “P < 0.05 versus respective baseline value: +P c 0.05 versus PAF alone at the respective time interval. Values are means ? SE.

The average time required for Ppa to increase above the baseline value was 26.6 f 5.3, 31.1 f 8.9, and 70.5 + 10.7 s in pigs treated with PAF, CGS 85 15 + PAF, and indomethacin + PAF (P < 0.05 versus other groups), respectively. The delayed onset of pulmonary hypertension in pigs treated with indomethacin + PAF cannot be explained by catheter length or dead space volume since we carefully controlled for these variables. Figure 5 shows that from 0.5-4 h, the PAF-induced pulmonary hypertension averaged -32 mmHg regardless of the presence of indomethacin or CGS 85 15. On the other hand, at 10 min indomethacin attenuated the PAFinduced increase in P,, by -4mmHg. For groups l-4. the baseline P,_,, averaged 6 mmHg and this was not significantly modified by any treatment (data not shown). Figure 6 shows that the sustained. PAF-induced increase in PVR was not significantly modified by CGS 85 15.

Fig. 4 Tracings of the initial mean pulmonary arterial pressures (P,,) in three pigs treated separately with PAF, indomethacin + PAF. or CGS 85 15 + PAF (curved arrows). The straight arrow indicates when a continuous infusion of PAF (10 ng/kg/min) was begun. Notice that indomethacin but not CGS 8515 delayed the initial onset (see curved arrows) of pulmonary hypertension and lessened the rate of rise in P,;, caused by PAF.

554

Prostaglandins Leukotrienes and Essential Fatty Acids

o-o e-0 A-A

O---o

Lyso PAP PAP Indomethacln + PAF CGS 8515 + PAP

Table 1 Effect of WEB 2086 on hemodynamics during a continuous infusion of PAF, indomethacin + PAF. and CGS 85 15 + PAF in pigs 0 Parameter

Time (min) 240 (Before WEB 2086)

250 (After WEB 2086)

Treatment

25 g

20

a 15

.o_o__o--o--o-o-Q-o-o

0

1

2

3

4

Hour Fig. 5 Effect of a continuous infusion of lyso PAF (n = 6), PAF (n = 7), indomethacin + PAF (n = 7) or CGS 8515 + PAF (n = 7) on mean pulmonary arterial pressure (Pr,). PAF and lyso PAF were infused at 10 ngkglmin. Values are means + SE. *P < 0.05 versus respective baseline value (0 h); +P < 0.05 versus PAF alone and CGS 8515 + PAF at the respective time interval.

o-o e-0 A-A 0-U

35??

30 --

3

25--

z

20--

$

15--

Lyso PAP PAP Indomethacin + PAP CGS 8515 + PAP

1o_~_o--o-o-+-% 0-l

:I 0

P,, (mmHg) Saline + PAF Indomethacin + PAF CGS 8515 + PAF

15.3tO.8 14.2 * 0.7 16.0 * 0.9

31.32 l.l* 29.1 f 1.5* 30.6 dz0.6*

15.2 + 0.9s 17.0 f 1.o* 14.7 f 0.68

PVR (mmHg/ml/sikg) Saline + PAF Indomethacin + PAF CGS 8515 + PAF

5.2 f 0.6 6.1 f 0.6 5.2 +_0.6

21.9 + 2.2* 30.4 f 3.0*+ 24.8 & 2.8*

8.0 + 0.3” 12.5 f 1.9*s 9.4 i: 1.38

ATPR (mmHg/ml/s/kg) Saline + PAF Indomethacin + PAF CGS 8515 + PAF

0 0 0

33.7 * 5.5* 13.7 f 5.8*§ 60.9 f 11.8*+ 40.7 f 9,8*#fi 16.0 + 3.1*+ 11.0 + 5.9*

ATPR, increase in total peripheral resistance compared to baseline (0 min) value. Values are mean + SE. For all treatment groups n = 7 prior to administration of WEB 2086. After WEB 2086, n = 3 for Saline + PAF and Indomethacin + PAF, and n = 5 for CGS 85 15 + PAF. *P < 0.05 compared to 0 min value. +P < 0.05 compared to Saline + PAF before treatment with WEB 2086; *P < 0.05 compared to Saline + PAF and CGS 8515 + PAF after treatment with WEB 2086; §P i 0.05 compared to the value obtained immediately before treatment with I mg/kg WEB 2086 (i.e. after infusion of PAF for 4: h). PAF was infused continuously at 10 ng/kg/min (IV).

concomitant with marked reductions in PVR. However, in indomethacin + PAF-treated pigs administered WEB 2086 (at 4 h), PVR remained significanlty above the 0 min value. WEB 2086 also decreased ATPR (i.e. the change in TPR relative to the 0 min value) in the three treatment groups. However, the ATPR values after treatment with WEB 2086 remained significantly greater in pigs treated with indomethacin + PAF as compared to saline + PAF or CGS 8515 + PAF.

I 1

2

3

4

Hour Fig. 6 Effect of lyso PAF, PAF, indomethacin + PAF, or CGS 8515 + PAF on pulmonary vascular resistance (PVR). *P < 0.05 versus respective baseline (0 h) value. +P < 0.05 versus PAF alone at the respective time interval. Values are means * SE.

whereas indomethacin slightly potentiated pulmonary vasoconstriction caused by PAF. Although lyso PAF was associated with a small increase in PVR at 4 h, we have observed similar results in pigs treated with saline. In the one pig treated with indomethacin + CGS 8515 + PAF, the sustained pulmonary and systemic hemodynamic findings were virtually the same as the mean values obtained in group 4, i.e. CGS 8515 + PAF (data not shown). Table 1 shows the effect of WEB 2086, a specific PAF receptor antagonist, on reversing PAF-induced alterations in hemodynamics (at 250 min) as compared to the 4 h value (i.e. immediately before administering WEB 2086). WEB 2086 completely reversed the increases in Pr,” observed in groups 24 and this occurred

Effect of PAF and blockade of cyclooxygenase S-lipoxygenase on bematologic parameters

and

Table 2 shows the effect of a continuous infusion of PAF on hematologic parameters, expressed as % baseline. For groups 1-4, the total WBC counts at 0 min were 20.1 f 3.6, 17.3 + 2.7, 14.5 f 1.9, and 20.6 ? 3.4 x 103/yl, respectively; for granulocytes the baseline values were 11.7 + 2.4, 9.8 f 2.4, 8.7 + 1.1, and 12.7 + 2.9 x 103/pl, respectively; for lymphocytes the baseline values were 7.9 f 1.5, 7.3 k 0.8, 5.5 k 1.0, and 7.5 f 0.8 x 103/p1, respectively. The 0 min platelet counts for groups l-4 were 446 + 46,434 + 51,443 f 46, and 487 f 43 x 103/ pl, respectively. None of the 0 min values were statistically significant among treatment groups. PAF caused modest leukopenia at 60-120 min that was not significantly modified by indomethacin or CGS 8515 (Table 2). Although PAF failed to induce statistically significant granulocytopenia at lo-240 min, significant lymphopenia and thrombocytopenia were evident during this time period (except at 60 min for platelets).

Effect of 5-Lipoxygenase

and Cyclooxygenase

Blockade on Porcine Hemodynamics

Table 2 Effect of a continuous hematologic parameters Parameter

infusion of PAF in the presence of indomethacin

Granulocytes (% baseline)

Lymphocytes (% baseline)

Platelets (% baseline)

Infusion of Platelet-Activating

Factor

555

or CGS 85 15 on porcine -~__

Treatment

WBC (?n baseline)

During Continuous

Lyso PAF PAF INDO + PAF CGS 8515 + PAF Lyso PAF PAF INDO + PAF CGS 8515 + PAF Lyso PAF PAF INDO + PAF CGS 8515 + PAF Lyso PAF PAF INDO + PAF CGS 8515 + PAF

Time (min) 0 10

60

120

180

100 100 100 100 IO0 100 100 100 100 100 100 100 100 100 100 100

96f 15 68 f 10* 65? 10* 66 f 14* 106f2’ 76? 16 72f 15 72 + 20 80 f 9 57 + 7* 54 + 9* 59 ? 7* 98 + 8 9orf 11 88 * IO* 89 f IO*

112f 13 68 + 10* 86+ 14 70+ I?* 131 + 1s 87f 17 105 it 20 79i 17 84& 17 43 + 1’: 60 & 12” 56 + 7” Y4 & 6 85 f 10” 81 7t 7* 86 + IO”

III f 77 f 105 f 76+ 136i99* 144 * 90+ 71 f 48 + 45 + 54 * 103 + 83i 78 * 82+

95* 18 78k 13 73 f 1I 74f 13* 98? 21 89f 18 75+ 15 76f 19 95k16 63*8* 67+ ll* 71 f lo* 99f 10 86 f lO* 89 ?r 8* 87 + lO*

3-w 10 8 13 I2 13 II 22 17 17 IO* 8* 3* 7 II* 7* IO*

1045 x 78 i- 7 100 + I.? 76 + IS* 126 + IU 107* II 12Y i_ 30 93 * 20 70+ I? 38 i- 7” 5.1 i 6’. 48 I 6..II1 + Ii 77 * Ii>‘76 I 7’ 791_ 10:”

Values are means f SE expressed as % baseline; n = 6 for lyso PAF and indomethacin (INDO) + PAF: n = 7 for PAF and CGS 8515 + PAF; WBC, total white blood cell count; *P < 0.05 compared with respective baseline value

Effect of lyso PAF, PAF, indomethacin + PAF, and CGS 8515 + PAF on plasma eicosanoids Figure 7 shows plasma TXB,, levels in groups l-4. PAF caused a rapid increase in plasma TXB? that was not attenuated by CGS 85 15. Indeed, at 4 h the plasma TXB, concentration was significantly greater in pigs treated with CGS 85 15 + PAF as compared to all other treatment groups. In contrast, indomethacin blocked the PAF-induced increases in plasma concentrations of TXB,. The plasma 6-keto-PGF,, concentrations in pigs treated with PAF (n = 4) or CGS 8515 + PAF (n = 7) were all below the detectable limit of 35 pg/ml plasma. The baseline (0 h) plasma LTB, concentrations for pigs treated with lyso PAF, PAF, indomethacin + PAF, or CGS8515+PAFwerel8-t14,23+6,5fl,and8+

o-o a-e A-A O-0

Lyso PAF PAF Indomethacin + PAF CGS 8515 + PAF

2 pg/ml, respectively. None of the plasma LTB, levels were significantly modified either within or between treatment groups.

DISCUSSION In a previous investigation, we reported that indomethacin blocked or markedly attenuated the hemodynamic effects of PAF administered as a bolus (2-20 “g/kg, IV) in pigs, indicating that cyclooxygenase products mediated the acute hemodynamic response ( 16). Similar findings have been reported by other investigators when PAF was infused as an IV bolus (5-7, 10-12). In the present study, we evaluated the effect of a continuous infusion of PAF (10 ng/kg/min) for 4 h on porcine hemodynamics in the presence and absence of indomethacin or CGS 8515 (5-lipoxygenase inhibitor). We were interested in exploring this phenomenon because the cardiovascular response to a sustained infusion of PAF is not well defined. This may be of clinical relevance since mechanisms relating to the actions of PAF likely depend on the manner in which the autocoid is tested ( I I).

Systemic hemodynamics

b_lY-_b

0-I

:!

0

h

)

: 1

2

3

J 4

Hour Pig. 7 Effect of PAF in the presence and absence of indomethacin or CGS 8515 and lyso PAF on arterial plasma levels of TXB?. Values jre means + SE as determined by RIA. *P < 0.05 compared to respective baseline (0 h) value; +P < 0.05 versus PAF alone at the respective time interval.

In the present investigation, PAF significantly increased TPR at 2.54 h and this effect was potentiated by indomethacin and blocked by CGS 85 15. Moreover, at 4 h the marked reduction in TPR in pigs treated with CGS 8515 + PAF was associated with significant systemic hypotension (i.e. mean aortic pressure 72 & 7 mmHg as compared to 115 +_ 9 and 116 + 8 mmHg for PAF and indomethacin + PAF, respectively). Thus, our findings regarding the sustained effect of PAF on the systemic circulation suggest that the autocoid importantly interacts with 5-lipoxygenase products in mediating in-

556

F’rostaglandins

Leukotrienes

and Essential Fatty Acids

creased TPR at 2.5-4 h. The importance of PAF-induced release of 5lipoxygenase products on the systemic circulation is further supported by our observation that CGS 8515 caused no attenuation of plasma concentrations of cyclooxygenase products. Indeed, the PAF-induced increase in plasma levels of TXBz were potentiated by the specific 5-lipoxygenase inhibitor at 4 h, yet the degree of systemic vasoconstriction was lessened (see Figs 3 and 7). Conversely, indomethacin potentiated PAF-induced systemic vasoconstriction (at 2.5-4 h) despite complete blockade of TXB2 biosynthesis. The apparent inactivity of TXA2 in modulating TPR may be related to its rapid conversion to the biologically inactive metabolite TXB,. After treatment with WEB 2086, the magnitude of the residual increase in TPR for pigs receiving indomethacin + PAF was significantly greater (at 250 min) as compared to pigs receiving PAF or CGS 85 15 + PAF (see Table 1). This further supports the hypothesis that a 5-lipoxygenase product(s) was responsible for the sustained contraction of systemic vessels. Thus, we propose that a continuous infusion of PAF triggers release of vasoactive 5-lipoxygenase products that predominantly affect the systemic circulation of pigs, and that this effect is potentiated by inhibiton of the cyclooxygenase pathway. Because LTB, is weakly vasoactive (17) and its plasma concentration was not elevated, it seems unlikely that LTB4 was involved in mediating the hemodynamic response. On the other hand, sulfidopeptide LTs (LTC4, LTD4, and LTE,) are powerful vasoconstrictors, especially in the porcine systemic circulation (IO, 18, 19), and they are products of the 5-lipoxygenase pathway (17). We have previously demonstrated that IV infusion of LTC, and LTD, in pigs predominantly affects the systemic circulation, and that the systemic effect is independent of cyclooxygenase products (19). Regarding attenuation of PAF-induced systemic vasoconstriction by CGS 85 15, our findings could be explained if the 5-lipoxygenase inhibitor also caused blockade of sulfidopeptide LT receptors. However, this seems unlikely since CGS 85 15 failed to modify LTC,-induced coronary vasoconstriction in isolated guinea pig hearts (14). Sulfidopeptide LTs are difficult compounds to accurately quantitate in biologic fluids since they are believed to be biosynthesized within a localized microenvironment and are rapidly catabolized (17). In other experimental systems, an important interaction appears to exist between PAF and 5-lipoxygenase products. For example, LTC, and LTD, were biosynthesized by porcine coronary arteries treated with l-100 nM PAF in vitro (20). PAF has been reported to induce release of sulfidopeptide LTs from isolatedperfused rat lungs (21, 22) and small intestine (23). In sensitized guinea pig lungs challenged with ovalbumin, the increased release of LTD, was blocked by BN 52021 (PAF receptor antagonist) (24). PAF-induced lethality in mice was reported to be dependent on endogenous

production of LTC, (25). Finally, we recently reported that exogenous infusion of cytokines (TNF, + interleukin1, (IL-l,)) in pigs induced systemic vasoconstriction that was blocked by CGS 8515 (15) or WEB 2086 (26). suggesting that cytokines caused release of PAF with subsequent generation of vasoconstrictor 5lipoxygenase products.

Pulmonary

hemodynamics

Indomethacin, but not CGS 8515, delayed the onset of PAF-induced pulmonary hypertension by -44 s (see Fig. 4), indicating that the initial increases (~2 min) in P,, were completely dependent on cyclooxygenase products. The inability of CGS 8515 to modify PAFinduced pulmonary vasoconstriction supports this argument. These results are consistent with previous reports which emphasize that cyclooxygenase products (primarily TXA,) mediate acute pulmonary vasoconstriction when PAF is infused as an IV bolus in pigs (3, 5-7, lO_ 12). The evidence for this is that indomethacin (3, 5, 10, ll), SQ 29548 (TXAI receptor antagonist) (5, 6) and OKY-046 (TXA, synthase inhibitor) (7, 12) blocked or markedly attenuated cardiovascular effects induced by PAF. In sheep, the early (O-15 min) increases in P,, and PVR induced by PAF (67 ng/kg/min over 15 min, IV) were blocked/attenuated by either indomethacin or meclofenamate (another cyclooxygenase blocker) concomitant with blockade of the increases in plasma levels of TXB, (27). Rabbits pretreated with the cyclooxygenase inhibitor ibuprofen or with TX synthase inhibitors (i.e. dazoxiben and OKY-046) are completely protected against lethal doses of PAF (15 pg/kg administered IV over 50-60 s), indicating that the cardiopulmonary dysfunction was primarily mediated by TXA2 (28). Thus, in sheep and rabbits the acute cardiovascular disturbances induced by PAF are also largely due to cyclooxygenase products. Since stimulation of PAF receptors may activate phospholipase A2 (1, 2) it is not surprising that administration of exogenous PAF results in release of vasoactive cyclooxygenase products. On the other hand, the rate of rise in Pr” was lessened by indomethacin after 2-5 min of PAF infusion (Fig. 4). Thus, the sustained PAF-induced pulmonary hypertension clearly becomes progressively less dependent on cyclooxygenase products with time. Indeed after 10 min, indomethacin failed to attenuate PAF-induced increases in P,, and PVR despite blockade of the increased plasma concentrations of TXB,. Our finding that CGS 8515 failed to modify PAF-induced increases in P,, and PVR at any time point suggests that 5lipoxygenase products, in contrast to their effect on the systemic circulation, also do not contribute to the pulmonary hemodynamic response. Thus, our results indicate that the sustained PAF-induced increases (after 10 min) in P,, and PVR are independent of both cyclooxygenase and 5-lipoxygenase products. This is further supported by our observation

Effect of SLipoxygenase

and Cyciooxygenase

Blockade on Porcine Hemodynamics

that indomethacin + CGS 8515 did not modify the in pulmonary sustained PAF-induced alterations hemodynamics. In the study reported by Burhop and coworkers (27), cyclooxygenase inhibitors blocked the early (~5 min) hypertensive response to a continuous infusion of PAF in sheep. This protective effect rapidly waned despite blockade of TXB, biosynthesis (27). Toyofuku et al (29) infused PAF into sheep at 200 ng/kg/min over 5 min (repeated at 4.5 min intervals). Except for the initial infusion of PAF, which was partially dependent on TXA,, the three subsequent infusions caused transient increases 111P,, and PVR that were independent of TXA, (29). The evidence for this was that OKY-046 (TXA? synthase inhibitor) failed to modify PAF-induced increases in P,, and PVR despite blockade of TXB, biosynthesis (73). Thus, taken together, these results indicate that the signal transduction mechanism utilized by PAF is dependent on the protocol utilized to administer the lipid autocoid. and that the cyclooxygenase pathway becomes less important in mediating the hemodynamic effects of PAF when the autocoid is continously infused or when the dose of PAF is increased. Because we previously demonstrated that infusion of PAF (20 ng/kg/min) in pigs pretreated with SRI 63-675 (PAF receptor antagonist) failed to elicit significant increases in PVR, it appears likely that the pulmonary hemodynamic response to PAF is specifically linked to PAF receptors (3, 16). Our finding that WEB 2086 reversed or markedly attenuated PAF-induced increases in P,,, and PVR at 4 h, in the presence and absence of indomethacin or CGS 85 IS, suggests that the sustained effect of PAF on the pulmonary circulation is mediated directly by PAF receptors on vascular smooth muscle. We cannot rule out, however, the possibility that potentiation of PAF-induced increases in PVR by indomethacin was secondary to enhanced biosynthesis of vasoactive S-lipoxygenase products. This is supported b!/ the fact that administration of WEB 2086 did not completely reverse the increased PVR value observed at 4 h in pigs treated with indomethacin + PAF (see T.ible 1 ).

Hematologic

findings

A continuous infusion of PAF caused modest and transient leukopenia (at 60-120 min), an effect that could be explained, interestingly, by lymphopenia but not granulocytopenia. The sustained lymphopenia (at lo240 min) and thrombocytopenia were not significantly modified by either indomethacin or CGS 8515. This suggests that the mechanism for these cellular changes was independent of cyclooxygenase and 5-lipoxygenase products thus supporting a direct effect of PAF on lymphocytes and platelets at 10-240 min. These results are in contrast to our earlier findings following a bolus infusion of PAF (20 ng/kg IV) (16). Under these conditions. PAF caused an early (at 30 s) leukopenia, granulo-

During Continuous

Infusion of Platelet-Activating

Factor

557

cytopenia, lymphopenia, and thrombocytopenia that was very transient in duration (~2 min). The PAF receptor antagonist SRI 63-675 completely blocked these hematologic changes ( 16). Moreover, the PAF-induced leukopenia, lymphopenia, and thrombocytopenia were blocked or markedly attenuated by indomethacin indicating that cyclooxygenase products mediated the early hematologic responses when PAF was infused as a bolus (16). Our finding that a continuous infusion of PAF failed to cause a sustained granulocytopenia is consistent with results reported in rabbits infused IV with PAF (83 ng/kg/min) for 2 h (30). For example, although acute neutropenia was present after 5 min, no significant neutropenia was evident at 1 and 2 h of PAF infusion (30). Taken together. these results suggest that neutrophils rapidly down-regulate during a continuous infusion of PAF. Although we did not assess the cell type(s) contributing to the release of 5-lipoxygenase products. it seems plausible that macrophages and circulating mononuclear and polymorphonuclear cells could be contributory. It is interesting to note, however, that in the present investigation, the most dramatic effect of PAF on circulating blood cells involved lymphocytes. Renkenon & coworkers (31) found that pretreatment of either lymphocytes or endothelial cells with PAF for 10 min in vitro enhanced lymphocyte binding to endothelial cell monolayers. Because PAF has profound effects on vascular endothelial and all inflammatory cells (including up-regulation of various adhesion molecules) (2. 3. 32), it seems likely that multiple cell-to-cell interactions could have contributed importantly to generation of S-lipoxygenase products in the present investigation.

Mechanism

of PAF action and clinical implications

Although a continuous infusion of PAF might be expected to down-regulate PAF-induced release of cyclooxygenase products, it does not appear that our infusion protocol caused down-regulation of all biochemical pathways involving the PAF receptor. The evidence for this is that PAF caused systemic vasoconstriction that was dependent of 5-lipoxygenase products at 2.5 4 h. Moreover, the specific PAF receptor antagonist WEB 2086 reversed PAF-induced pulmonary hypertension at 4 h (see Table 1). These would not be the expected findings if all PAF receptors were downregulated. Thus, with regard to the, physiological effects of PAF on systemic and pulmonary hemodynamics, a sustained infusion of the autocoid does not lead to tachyphylaxis. Similar observations have been reported by other investigators ( 11, 30). We believe our results are of potential clinical relevance with regard to therapy of diseases involving release of PAF. For example. those pathophysiological situations in which biosynthesis of PAF is intermittent and of short duration may respond well to cyclooxygenase blockade. On the other hand. clinical mala-

558

Prostaglandins

Leukotrienes

and Essential Fatty Acids

dies involving sustained release of PAF might be refractory to (or worsened by) cyclooxygenase blockade and thus require alternate therapy, e.g. a 5lipoxygenase inhibitor or PAF receptor antagonist. The results of the present investigation and our previous work, which demonstrated that pretreatment of pigs with a PAF receptor antagonist blocked cardiopulmonary effects of PAF (16), indicate that PAF influences porcine hemodynamics by at least three different mechanisms that are receptor linked. First, the early (~10 min) PAF-induced pulmonary hypertension is dependent on cyclooxygenase products. Second, the later (2.5-4 h) PAF-induced increases in TPR are dependent on 5lipoxygenase products. And third, PAF apparently modulates vascular resistance of both pulmonary and systemic circulations through a direct receptor-dependent mechanism (unrelated to cyclooxygenase and 5lipoxygenase products). The evidence for the latter is the rapid decline in PVR and TPR caused by WEB 2086 in pigs infused with PAF, CGS 8515 + PAF, or indomethacin + PAF. This mechanism is consistent with the hypothesis that PAF receptors may be coupled directly to voltage-regulated calcium channels (2). Alternatively, PAF may increase the level of intracellular calcium in vascular smooth muscle cells through receptor-mediated stimulation of phospholipase C-dependent hydrolysis of phosphatidylinositol-4, 5-bisphosphate (1, 2). This would result in generation of the second messengers inositol trisphosphate and diacylglycerol, which mobilize internal stores of calcium and activate protein kinase C, respectively (1, 2).

Acknowledgments The authors thank Dr John Devlin (Boehringer Ingelheim Corp., Ridgefield, CT) for providing WEB 2086; and Dr Richard Love11 (Ciba-Geigy Corp., Summit, NJ) for providing CGS 85 15. This work was supported by National Heart, Lung, and Blood Institute Grants HL-32726 (NCO) and 5-F32HL-07985 (KTK-E).

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Effect of 5-Lipoxygenase

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29. Toyofuku T, Kobayashi T, Koyama S. Kusama S. Pulmonary vascular response to platelet-activating factor in conscious sheep. Am J Physiol 1988; 255 (Heart Circ Physio124): H434-H440. 30. Deavers S I. Arroyave J M, Prihoda T J, McManus L M. Cardiopulmonary and intravascular alteration during the sustained infusion of PAF. J Lipid Med 199 1: 4: 135-l63. 3 1. Renkonen R, Mattila P, Turunen J-P. Hayry P. Lymphocyte binding to and penetration through vascular endothelium is stimulated by platelet-activating factor. Stand J Immunol 1989; 30: 673-678. 32. Braquet P. Rola-Pleszczynski M. Platelet-activating factor and cellular immune responses. Immunol Today 1987: 8: 3455352.

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